ABSTRACT Erythrocytes (red blood cells; RBCs) and their progenitors express distinct proteins, which underlie their unique biology, and which provide a molecular basis for many blood diseases, including diverse anemias, such as those arising during renal failures as a result of low red blood cell production and lifespan. Importantly, mammalian RBCs lack nuclei and other major organelles, and hence neither transcriptional profiling by RNA- sequencing nor recombinant transfection?powerful techniques in other cell types?can be used to reveal RBC gene functions and pathways. Proteomics methods, in contrast, allow for a detailed analysis of RBC proteins, and pioneering studies have revealed that RBCs, while dominated by hemoglobin (98%), express on the order of 1,500 to 2,000 distinct proteins. Many of these proteins play critical roles in erythrocyte function, including key metabolic and bioenergy roles, and cytoskeletal roles in controlling RBC cell shape. More than 500 proteins in RBCs are of entirely unknown function. A fundamental question in RBC biology is thus how all of these proteins work together to support proper RBC function and development. Building deep mechanistic understanding of RBC biology requires accurately delineating the precise membership of protein complexes specific to RBCs, as these carry out key functions unique to these cells. We propose to perform the first systematic, global exploration of native protein-protein interactions (PPIs) in RBCs, using a powerful new technology to examine those interactions directly among endogenous proteins in human and other mammalian RBCs. Our proposed experiments combine protein biochemistry, quantitative mass spectrometry proteomics and integrative computer modeling to reliably define the extended PPI networks and multiprotein complexes native to RBCs, helping to lay rich new mechanistic foundations for interpreting RBC biology. By the end of this grant, we will have performed nearly 2,000 mass spectrometry experiments on native protein complexes isolated from primary RBCs and their progenitors, defining the RBC interactome, including both shared and novel protein complexes, to an unprecedented degree. As a result of this work, RBCs will be the first primary human cell type with a near complete map of stable protein complexes, giving new insights into erythrocyte biology and development, and laying the foundation for future attempts to intervene, chemically or genetically, in diseases affecting these critical cells.